We propose to elucidate the primary structure of band 3, the predominant polypeptide of the human erythrocyte membrane. This component comprises roughly 25% of the membrane protein mass, contributing approximately 1 x 10 to the 6th power copies per cell. It spans the membrane asymmetrically; its very hydrophobic behavior on isolation appears to reflect this adaptation for membrane insertion. It is a glycoprotein with its carbohydrate residues facing outside the cell. At its cytoplasmic pole, it has a binding site (or sites) for at least two glycolytic enzymes. Its function appears to be the facilitated diffusion of anions (chloride and bicarbonate in particular) across the membrane. It is our aim to correlate its structure with function in the context of its membrane integration. We have cleaved this polypeptide in situ to generate fragments which represent the outer-surface, cytoplasmic surface, and membrane-spanning regions. Two of these pieces, representing nearly half the molecule, have been purified at the preparative level. (They are a 17,000 dalton membrane-spanning piece and a 23,000 dalton fragment from the cytoplasmic end of band 3). Tryptic sub-fragments of these pieces are now being prepared for sequence analysis. Procedures are now being developed for preparing complementary fragments, representing all or nearly of the remainder of the band 3 polypeptide. Special techniques have been devised or adapted for handling the insoluble hydrophobic fragments (e.g., a novel preparative gel electrophoresis apparatus, a two-phase partition chromatography system and chemical modifications to increase peptide solubility). We are now in a strong position to carry out the amino acid sequence analysis of the band 3 polypeptide.